The present invention is related to a composition and method for regulating the adhesion of cells, organisms, and molecules to hydrophobic surfaces. More specifically, the present invention is directed to a biomolecule, such as proteins, peptides amino acids, nucleic acids, lipids, and carbohydrates conjugated to end-group activated polymers (EGAPs) and uses thereof.
Normal development and function in living organisms require interactions between cells and the molecules in the surrounding environment. One way cells communicate is via molecules that span the membrane of the cell called transmembrane proteins. When the portion of the transmembrane protein which is outside of the cell encounters specific molecules in the surrounding environment, it undergoes structural and conformational changes which triggers biological reactions inside the cell.
For example, in vivo, cells form complex multilayer structures which ultimately form tissues and organs. Tissue and organ formation, however, requires specific contacts with the environment. These cells are referred to as xe2x80x9canchorage-dependentxe2x80x9d because they will not grow properly, if at all, unless they are anchored to others cells, an extracellular matrix (ECM), or other surface.
An ECM is a complex and variable array of molecules secreted by cells, such as collagens, glycosaminoglycans, proteoglycans, and glycoproteins. Together these cellular products form the basal lamina, bone, and cartilage which give tissues and organs their shape and strength. In fact, contact between anchorage-dependent cells and the ECM in many instances plays a dramatic role in determining the cells"" shape, position, metabolism, differentiation and growth.
Cell contact is also important in other biological functions, such as the activation of an immune response. The immune system is a complex network of cells that have the ability to recognize and rid the body of foreign substances, such as viruses, bacteria and parasites. One mechanism used by the immune system to rid itself of foreign substances is a humoral response. A humoral response involves activation of specific cells called B cell lymphocytes. B-cells are activated when transmembrane proteins on their surface bind to foreign substances called antigens. Specifically, binding of B-cells to antigens stimulates B cells to proliferate and differentiate into immunoglobulin or antibody producing plasma cells.
The antibodies produced by plasma cells travel throughout the body binding to the pathogen or foreign substance. Binding of antibodies to foreign substances activates several other immunological pathways, including the xe2x80x9ccomplementxe2x80x9d pathway. The complement pathway is designed to destroy the foreign substance and to initiate an inflammatory response in the organism.
While cell contact with other cells and the environment is critical to the overall health and biological function of an organism, it creates unique problems in the art of biotechnology. Specifically, two areas where cell contact requirements create problems are: (1) cell culture; and (2) biomaterial transplantation.
Tissue or cell cultures comprise cells from a plant or animal which are grown outside the organism from which they originate. These cells are often grown, for example, in petri dishes under specific environmental conditions. Cell cultures are of great importance because they represent biological xe2x80x9cfactoriesxe2x80x9d capable of producing large quantities of biological products such as growth factors, antibodies, and viruses. These products can then be isolated from the cell cultures and used, for example, to treat human disease. In addition, cell cultures are a potential source of tissue which could be used for transplantation into humans. For example, cell cultured skin cells could potentially be used in skin grafts to replace diseased or damaged skin. Finally, cell cultures usually comprise cells from only one or a few tissues or organs. Consequently, cell cultures provide scientists with a system for studying the properties of individual cell types without the complications and risk of working with the entire organism. For example, the effects of pharmaceutical drugs on certain cell types could be tested on cell cultures prior to clinical trials in order to assess the drug""s health risks.
Like most cells in vivo, cells grown in culture are either anchored to an ECM or another cell. Only cells of the circulatory system (e.g., lymphocytes and red blood cells) grow unattached and suspended in solution in vitro. Many anchorage-dependent cells can grow on glass or plastic surfaces, such as polystyrene. These cells, however, often lose their natural architecture and do not function normally (e.g., the ability to differentiate and respond to hormones). Accordingly, these cells do not precisely mimic a cell""s biological functions in vivo and thus have limited potential.
For this reason, glass and plastic cell culture dishes are often coated with an ECM protein such as collagen, fibronectin, laminin and the like. These proteins bind to surfaces such as polystyrene through a process known as adsorption. Although ECM coated cell culture surfaces have led to improved culture conditions, they are far from ideal.
First, biomolecules, such as proteins, often become inactivated upon adsorption to hydrophobic surfaces. The biological activity of proteins is conferred by their unique structure and their ability to undergo conformational changes upon binding to a substrate or other physiological event. In one study, the structure of proteins was measured using a technique called microcalorimetry. Microcalorimetric studies demonstrated that proteins which are bound to hydrophobic surfaces loose essentially all their cooperatively folded structure compared to the same protein in solution. Because a protein""s structure and its ability to undergo conformational changes strongly correlates with biological activity, these data suggest that most proteins that are adsorbed by a hydrophobic surface loose there in vivo biological activity.
Second, the conformation and orientation of immobilized proteins have important effects on the nature of their interaction with cells. D. J. Juliano, S. S. Saaedra and G. A. Truskey, Journal of Biomedical Materials Research 2-7 1103-1113 (1993). Both are influenced by the chemistry and physical properties of the underlying substrate as well as by the method of immobilization. K. Lewandowska, E. Pergament, N. Sukenik and L. A. Culp, The Journal of Biomedical Materials Research 21 1343-1363 (1992).
Third, like in vivo, cells in culture release molecules such as serum proteins and growth factors into the culture media. As discussed above, the secretion and concentration of these molecules in the culture media are critical to the biological function of neighboring cells. Under current cell culture conditions, the careful balance and concentration of secreted molecules are disrupted because secreted molecules are adsorbed by the cell culture surface. Thus, the communication and biological function of cells grown under current cell culture techniques does not mimic in vivo environment.
Finally, the surface concentration of ECM components is a critical factor in the regulation of cell behavior. The ability to control and vary surface biomolecule concentration is therefore of upmost importance and depends on the method of immobilization and in some cases the physical nature of the base material. Simple ECM adsorption to cell culture substrates does not meet these requirements.
In short, to date there is no single method for conjugating proteins to potential cell culture substrates which addresses all these major concerns. Thus, current research is hindered by the fact that cell cultures do not accurately mimic an in vivo environment.
A second problem area created by cell contact is biocompatibility. It is generally acknowledged that artificial biomaterials, including fabricated biomedical polymers, are much less immunologically active than transplants or tissue-derived biomaterials. Nevertheless, the use of non-physiological biomaterials in many lifesaving medical devices, either extracorporeal or implanted, often leads to adverse side-effects for the patient.
The adverse side-effects observed are usually a consequence of contact between cells, proteins, and other biological fluids in the blood with the artificial biomaterial. Typically, contact with the artificial biomaterial activates two major biological processes: coagulation and complement. As discussed above, the complement pathway is designed to destroy the foreign substance and to initiate an inflammatory response in the organism.
Activation of the coagulation cascade can be controlled to a limited extent with the use of anticoagulants, e.g., heparin. Heparin, however, is not well suited for extended use such as in the case of a permanent implant. Further, currently there is no clinically available agent that can prevent or suppress artificial surface-initiated activation of complement. Thus, activation of the coagulation and complement systems upon blood contact is a major problem with respect to biomaterial transplantation.
From the foregoing, it will be appreciated that it would be an advancement in the art to provide a method of coating tissue culture surfaces with ECM proteins or other biomolecules that does not destroy the biological activity of the biomolecule.
It would also be an advancement in the art if the biomolecule coated surface could be used to adhere prokaryotic and eukaryotic cells, viruses, and other molecules for the purpose of biological assay.
It would be a further advancement in the art if the tissue culture cells could adhere and grow on the biomolecule coated surface.
It would be yet another advancement in the art if the biomolecule coated surface did not adsorb proteins and other molecules secreted by the cells in culture.
Finally, it would be an-advancement in the art if biomaterial used in transplantation could be coated with an immunologically inert biomolecule to prevent or minimize host rejection.
Such compositions and methods are disclosed and claimed herein.
The present invention is directed at a composition and method for regulating the adhesion of cells and biomolecules to hydrophobic surfaces and hydrophobic coated surfaces. Generally, the composition is an end-group activated polymer (EGAP) generally comprises a block copolymer surfactant backbone and an activation or reactive group. The polymeric block copolymer surfactant of the present invention may be any surfactant having a hydrophobic region capable of adsorbing onto a hydrophobic surface and a hydrophilic region which extends away from the surface when the hydrophobic region is adsorbed to the hydrophobic surface. In one embodiment, the EGAP is synthesized by reacting the block copolymer surfactant with 4-nitrophenylchloroformate followed by 2-(2-pyridyldithio)ethylamine.
A large range of biomolecules can be conjugated to EGAP, include natural or recombinant growth factor, mitogens, growth peptides, differentiating factors, sugars, carbohydrates, polysaccharides, lipids, sterols, fatty acids and nucleic acid. In one embodiment, the biomolecule contains a natural or artificial thiol group. These biomolecules are conjugated to EGAP via a disulfide linkage.
The biomolecule conjugated EGAP surface can be put to a wide variety of uses. For example, the composition can be used to attach organisms and molecules for growth or biological analysis. Briefly, this is done by contacting a hydrophobic surface with an EGAP for a time sufficient for the EGAP to be adsorbed by the hydrophobic surface. A biomolecule is then conjugated to the EGAP adsorbed to the hydrophobic surface to form a biomolecule conjugated EGAP surface. After washing of unconjugated biomolecule, organisms or molecules are placed in contact with the biomolecule conjugated EGAP coated surface such that the organism or molecule adheres to the biomolecule conjugated EGAP coated surface. In one embodiment, the organism or molecule is a eukaryotic or prokaryotic cell, a virus, an antibody or a pharmaceutical drug.
The biomolecule conjugated EGAP surface can also be used to selecting at least one desired organism or molecule from a mixture of at least two organisms or molecules. This is done by first adsorbing EGAP onto a hydrophobic surface. A biomolecule unique for a desired organism or molecule being selected is then conjugated to the EGAP adsorbed to the hydrophobic surface. A mixture of organisms or molecules containing the desired organism or molecule is then contacted with the biomolecule conjugated EGAP coated surface and the desired organism or molecule is allowed to adhere to the unique biomolecule. Finally, non-adhered organisms or molecules are removed.
These and other objects and advantages of the present invention will become apparent upon reference to the accompanying drawings and graphs and upon reading the following detailed description and appended claims.